ABSTRACT
The peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-activated transcription factor and a key regulator of lipid homeostasis. Numerous fatty acids and eicosanoids serve as ligands and activators for PPARalpha. Here we demonstrate that S-hexadecyl-CoA, a nonhydrolyzable palmitoyl-CoA analog, antagonizes the effects of agonists on PPARalpha conformation and function in vitro. In electrophoretic mobility shift assays, S-hexadecyl-CoA prevented agonist-induced binding of the PPARalpha-retinoid X receptor alpha heterodimer to the acyl-CoA oxidase peroxisome proliferator response element. PPARalpha bound specifically to immobilized palmitoyl-CoA and Wy14643, but not BRL49653, abolished binding. S-Hexadecyl-CoA increased in a dose-dependent and reversible manner the sensitivity of PPARalpha to chymotrypsin digestion, and the S-hexadecyl-CoA-induced sensitivity required a functional PPARalpha ligand-binding pocket. S-Hexadecyl-CoA prevented ligand-induced interaction between the co-activator SRC-1 and PPARalpha but increased recruitment of the nuclear receptor co-repressor NCoR. In cells, the concentration of free acyl-CoA esters is kept in the low nanomolar range due to the buffering effect of high affinity acyl-CoA-binding proteins, especially the acyl-CoA-binding protein. By using PPARalpha expressed in Sf21 cells for electrophoretic mobility shift assays, we demonstrate that S-hexadecyl-CoA was able to increase the mobility of the PPARalpha-containing heterodimer even in the presence of a molar excess of acyl-CoA-binding protein, mimicking the conditions found in vivo.
Subject(s)
Acyl Coenzyme A/pharmacology , Coenzyme A/pharmacology , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Acyl-CoA Oxidase , Animals , Cell Line , Chromatography, Affinity , DNA-Binding Proteins/drug effects , Dimerization , Genes, Reporter , Glutathione Transferase/genetics , Histone Acetyltransferases , Ligands , Mice , Models, Molecular , Nuclear Receptor Coactivator 1 , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Protein Biosynthesis , Protein Conformation , Rats , Receptors, Cytoplasmic and Nuclear/drug effects , Receptors, Retinoic Acid/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Retinoid X Receptors , Spodoptera , Trans-Activators/metabolism , Transcription Factors/drug effects , Transcription, Genetic , TransfectionABSTRACT
We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The determined molecular masses are often sufficient for identification. If not, the proteins are subjected to mass spectrometric peptide mapping followed by database searches. Apart from protein identification, the protocol also yields information on posttranslational modifications. The protocol was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA.
